Throughout this application, various publications are referenced in parentheses and citations provided for them. The disclosure of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains.
Retroviruses appear to be the method of choice as vectors for the transfer of exogenous genes into humans. In particular, the cloning, transfer, and expression of human globin genes into erythroid cells in culture has raised the possibility of autotransplantation of bone marrow cells with normal .beta.-globin genes as an approach to the therapy of .beta.-thalassemia and sickle cell anemia in humans (1). Retroviral vectors are the most efficient means of transferring genes into cells. This high efficiency is a requirement for experiments whose goal is human globin gene therapy because only a limited number of bone marrow stem cells can be obtained, and as many as possible must acquire and express the transferred genes to ensure repopulation of sufficient marrow elements to produce normal amounts of hemaglobin.
A major prerequisite for the use of retroviruses is to insure the safety of their use (2). The major danger of the use of retroviruses for gene therapy is the possibility of the spread of wild-type retrovirus in the cell population. The proliferation of wild-type virus can lead to multiple integrations of the retrovirus into the genome which may result in the activation of potentially harmful genes such as oncogenes (3, 4). The development of packaging cell lines that produce only replication-defective retroviruse:; has increased the utility of retroviruses for gene therapy (5-9). In these cell lines, the sequence required for packaging of the viral RNA (.psi. sequence) bias been deleted, therefore the packaging cell produces viral proteins but is unable to package the viral RNA genome into infectious virions. When these packaging lines are transfected with a replication-defective retroviral vector containing an intact .psi. sequence required for its own packaging, wild-type retrovirus has been shown to arise (6, 10, 11) presumably due to recombination events between the helper virus genome and the vector virus. For example, high titer amphotropic retroviral stocks generated by transfer of a defective neomycin-containing retrovirus into the amphotropic packaging cell line PA12 (containing the .psi. deletion) have been shown to produce infectious amphotropic helper virus (10, 11). To circumvent this problem, additional mutations have been made in the defective virus of newer helper cell lines (11). These have included deletions in the 3' LTR of the helper virus component, and additional deletions of portions of the 5' LTR as well. One of these defective amphotropic constructs has been used to produce a retroviral packaging line, PA317, that has recently been reported to eliminate wild type retrovirus production after retroviral transfection. However, using this packaging line, two recombinational events could still produce intact retrovirus. Cell lines containing both 3' and 5' LTR deletions as well as the packaging mutation were also constructed but were not useful because of the relatively low titers obtained with these constructs.
We have created novel ecotropic and amphotropic retrovirus packaging cell lines which should virtually eliminate the possibility of recombination between the helper virus and the vector virus leading to wild-type retrovirus. In our cell lines, the helper virus DNA has been separated onto two plasmids; the gag and the pol genes are on one plasmid and the env gene is on another plasmid. In addition, the packaging sequence and the 3' LTR have been deleted in both plasmids. With this type of strategy at least three recombination events between the two helper plasmids and the vector virus are necessary to generate a wild-type virus. We will describe the development of both a stable ecotropic and a stable amphotropic packaging line that are both efficient and safe for use in gene transfer experiments. An ecotropic packaging line concerns a virus which can only infect or transfect cells of the same species. In an amphotropic packaging line, the virus can infect or transfect a wide range of host cell species.